DESCRIPTION (APPLICANT'S ABSTRACT): Degradation of type I membrane proteins involves their extraction from the membrane, transfer to the cytosol, and destruction by the proteasome. This pathway is exemplified by the degradation of MHC class I molecules in cells that express the HCMV US2 or US11 products, both of which dramatically enhance the rate of degradation. We shall develop a chemistry-based approach to dissect this pathway in more detail. The generation of a chemically charged suppressor tRNA, equipped with a cleavable, photoreactive radiolabeled amino acid derivative should allow site-specific incorporation of the photoprobe into protein in membrane-supplemented in vitro translation systems. Photocrosslinking and subsequent reduction should result in transfer-of-label and may identify interacting partners in the US2-mediated degradation pathway of MHC Class I molecules. The next step in degradation is the removal of N-linked glycans by a peptide N-glycanase, of unknown identity and most likely confined to the cytosol. As possible inhibitors of this activity, electrophilic derivatives of G1cNAc will be prepared, also in radiolabeled or biotinylated form to assist in identification and purification of the target enzymes for which a novel assay has been developed. This reaction is followed by conjugation of the substrate with ubiquitin (Ub) to target the substrate to the proteasome. Prior to proteasomemediated proteolysis, the Ub moieties must be removed in a reaction catalyzed by members of the Ubisopeptidase multigene family, about whose specificity and control relatively little is known. We have synthesized an Ub-vinylsulfone that acts as an irreversible, covalent inhibitor of at least some of these isopeptidases. This class of compounds will be developed further to probe the in vitro dislocation of MHC Class I molecules. Proteolysis, finally, is carried out by the proteasome. We shall synthesize proteasome substrates that are heterobifunctional compounds containing a photoreactive moiety and an electrophile, to probe more accurately interactions of substrates -and potentially cellular proteins- with the proteasome. In the aggregate, the resulting tools are expected to shed new light on this poorly understood pathway of membrane protein turnover. The chemical-biological approach should allow development of more widely applicable molecular probes that could be used to manipulate their targets in health and disease.